The specific aims of this proposal are to explore a role for reactive oxygen species in the carcinogenesis of dioxin-like agents, and to determine whether cytochrome P450-catalyzed oxidative estrogen metabolism is in part responsible for the gender differences that these agents display with respect to their carcinogenic potencies. In order to accomplish these objectives, we are proposing to integrate the following laboratory analyses into the ongoing exposures studies of the National Toxicology Program, which are being performed in female animals, as well as perform a parallel series of experiments in male animals that we are currently exposing to the same polychlorinated biphenyl agents under separate auspices. The following experimental end-points will be measured in liver tissues: 1) microsomal CYP1/2 P450 activities by enzymatic substrate assays; 2) microsomal estrogen catechol metabolism by gas chromatography-electron capture detection; 3) production of oxygen free radicals by electron paramagnetic resonance/spin trap analyses; and 4) oxidative DNA damage by gas chromatography-mass spectrometry detection of oxidized DNA base adducts. The comparisons of male and female responses to dioxin-like agents will provide further elucidation of the biological pathways responsible for their carcinogenesis, and the comparisons of the P450-catalyzed metabolism of estrogen and concomitant production of oxygen free radicals will provide further evidence for a role of the Ah receptor in a mechanistic pathway of carcinogenesis. These data should prove useful in assessing the utility of the Toxic Equivalency Factor approach for predictive modelling of the carcinogenicity of dioxin-like mixtures, as well as in the identification of other possible risk factors for individuals exposed to dioxin-like contaminants.